Development of a novel dehydrogenase and a stable cofactor regeneration system

Consensus sequence;Glucose dehydrogenase;Cofactor regeneration;Protein engineering;Salt and solvent stability;Biocatalysis

Vázquez-Figueroa, Eduardo ; Chemical Engineering

University:Georgia Institute of Technology

Department:Chemical Engineering

关键词: Consensus sequence;    Glucose dehydrogenase;    Cofactor regeneration;    Protein engineering;    Salt and solvent stability;    Biocatalysis;   

Others  :  https://smartech.gatech.edu/bitstream/1853/31685/1/vazquez-figueroa_eduardo_200812_phd.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

The first goal of this work focused on the development of an amine dehydrogenase (AmDH) from a leucine dehydrogenase using site-directed mutagenesis.We aimed at reductively aminating a prochiral ketone to a chiral amine by using leucine dehydrogenase (LeuDH) as a starting template.This initial work was divided into two stages.The first focused mutagenesis to a specific residue (K68) that we know is key to developing the target functionality.Subsequently, mutagenesis focused on residues known to be in close proximity to a key region of the substrate (M65 and K68).This approach allowed for reduced library size while at the same time increased chances of generating alternate substrate specificity.An NAD+-dependent high throughput assay was optimized and will be discussed. The best variants showed specific activity in mU/mg range towards deaminating the target substrate. The second goal of this work was the development of a thermostable glucose dehydrogenase (GDH) starting with the wild-type gene from Bacillus subtilis.GDH is able to carry out the regeneration of both NADH and NADPH cofactors using glucose as a substrate.We applied the structure-guided consensus method to identify 24 mutations that were introduced using overlap extension.11 of the tested variants had increased thermal stability, and when combined a GDH variant with a half-life ~3.5 days at 65℃ was generated--a ~10⁶increase in stability when compared to the wild-type.The final goal of this work was the characterization of GDH in homogeneous organic-aqueous solvent systems and salt solutions.Engineered GDH variants showed increased stability in all salts and organic solvents tested.Thermal stability had a positive correlation with organic solvent and salt stability.This allowed the demonstration that consensus-based methods can be used towards engineering enzyme stability in uncommon media.This is of significant value since protein deactivation in salts and organic solvents is not well understood, making a priori design of protein stability in these environments difficult.